Journal of General Microbiology (1982), 128, 381-385.
Printed in Great Britain
38 1
Far Ultraviolet Light Sensitive Derivatives of Streptomyces clavuligerus
By G . S A U N D E R S * A N D G . H O L T
School of Engineering and Science, The Polytechnic of Central London, 11 5 New Cavendish
Street, London WlM 8JS, U.K.
(Received 19 December 1980; revised 21 Ma-y 1981)
Five far ultraviolet light sensitive (Uvs) strains - CL77, CL89, CL90, CL104 and CL105 were isolated from an auxotrophic strain of Streptomyces clavuligerus (ATCC 27064). When
compared to their progenitor, strain CL7, all exhibited a changed mutagenic response to far
ultraviolet light (FUV). Strains CL77 and CL104 failed to yield detectable mutants, whereas
induced mutants were obtained in strains CL90 and CL89, but only at low FUV doses (up to
20 J m-2). Strain CL89 was hypermutable at these low doses relative to strain CL7. Strain
CL105 gave an increased response in terms of mutants per surviving cell. Caffeine increased
the FUV-induced mutation frequency at a particular FUV dose in strain CL7 by up to
3000%. All the Uvs strains showed an altered response to caffeine compared with strain
CL7.
INTRODUCTION
DNA repair may be considered as an obligate step in the process of ‘indirect’ mutation
induction intercalated between production of a pre-mutational lesion and realization of a
mutant clone. By interfering with DNA repair, either by chemical inhibition of repair enzymes
or mutation of DNA repair genes, one may in turn affect the process of mutation induction.
The first radiation-sensitive (i.e. genetically repair-deficient) mutant was isolated by Hill
(1958) from Escherichia coli. Since then the number of such mutants has steadily grown. In
E. coli, for example, there are at present some 25 loci known to be involved in DNA repair of
damage induced by far ultraviolet light (FUV), and in all of these the mutagenic response to
FUV, and in certain cases to other mutagens, is altered (Kimball, 1978).
Kondo et al. (1970) have studied the response of FUV-sensitive (Uvs) strains of E. coli to
different mutagens. They found that rec strains, which failed to yield mutants in response to
FUV and ionizing radiations, were mutated by the alkylating agent N-methyl-”-nitroN-nitrosoguanidine (NTG). However, methyl methanesulphonate (MMS), another alkylating
agent, followed the pattern of FUV and ionizing radiations in that no MMS-induced mutants
were obtained in rec strains. Previously, Witkin (1967) had reported that exr strains were
mutated normally by NTG.
The secondary metabolites of actinomycetes provide the majority of industrially and
agriculturally important antibiotics (Berdy, 1974). Strains used industrially will usually be
subjected to a programme of strain selection and improvement, in which a clear insight into
the mechanism of mutation induction in the particular strain under investigation would be
useful.
Streptomyces clavuligerus has been shown to produce a new antibacterial agent, clavulanic
acid (Reading dz Cole, 1977), and in the present study we have tested the response of Uvs
derivatives of S . clavufigerus to different mutagens including FUV, ionizing radiations and
alkylating agents. Additionally, the effect of caffeine on FUV-induced mutation frequencies
has been investigated in both repair-deficient and repair-proficient strains.
0022-1287/82/0000-9695 $02.00
O 1982 SGM
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382
G. S A U N D E R S A N D G . H O L T
METHODS
Media. Yeast extract agar (YEA) and purified agar (PA) were from Oxoid. Yeast extract-malt extract agar
(YM) was as described in Shirling & Gottleib (1966). All media were prepared at pH 7.2.
Organism and cultural conditions. Streptomyces clavuligerus 27064N, a subculture of strain ATCC 27064,
was obtained from Beecham Pharmaceuticals and maintained on YM.
Isolation of Uvs strains. A spore suspension of a hypoxanthine-requiring auxotroph of S. clavuligerus 27064N
was irradiated with FUV to give approximately 20% survival (48 J mU2).Survivors were spread on YEA plates
and examined for FUV sensitivity using the technique devised by Harold & Hopwood (1970).
Preparation of spore suspensions. Spores were harvested from 14-21 d old cultures grown at 26 "C on YM
slopes and resuspended in 0.01% (v/v) Tween 80 (Hopkin & Williams) by mixing on a vortex blender together
with glass beads. Suspensions were then filtered through sintered glass (Fisons, porosity 1). Spore suspensions
were prepared to give approximately 4 x lo8spores ml-l.
Mutagenic treatments. A Hanovia bactericidal FUV lamp giving 95 96 of its output at 254 nm was used. Dose
was measured using a 'Blak-ray' meter (Ultra-violet Products, San Gabriel, Calif., U.S.A.). Spore suspensions were
irradiated in 1.5 ml quantities in glass Petri dishes of 3 cm diameter. During irradiation the spore suspension was
agitated by a magnetic stirrer.
A Siemans Dermoplan therapy unit generating X-rays of 50 kV (250 mA), with the beam directed vertically
into an open Universal bottle containing 0.5 ml of sample, was also employed. The calculated dose rate was
537 rad min-'.
Methyl methanesulphonate (MMS) treatment was carried out in phosphate buffer pH 7. Spores suspended in
Tween 80 were sedimented and resuspended in buffer. MMS was added and after 15 min spores were once more
sedimented, washed, and then resuspended in Tween 80. MMS concentrations used ranged from 0.05 to 0.2%
(v/v). N-Methyl-N'-nitro-N-nitrosoguanidine
(NTG) treatment was carried out in the same way except that citrate
buffer, pH 5.6, was used. NTG concentrations used ranged from 0.5 to 3.0 mg ml-l.
Assay for mufagenesis. After mutagenic treatment, 0.1 ml samples of undiluted spore suspension were mixed
with 10 ml of soft YEA (0.8 % agar) (layer 1) and poured into a Petri dish. After allowing time for the agar to set,
a second 10 ml layer (layer 2) of soft agar was poured on top. Plates were then incubated for 20 h at 26 O C , a time
selected after experimentation as the optimum time to obtain expression. At the end of this period, a third (layer 3)
10 ml layer of PA containing either acriflavine or rifampicin was added. The final concentrations of acriflavine and
rifampicin in a plate were 50 pg ml-' and 1 pg ml-I, respectively. Plates were incubated at 26 O C for 7 d and then
scored for mutants. Survival plates were prepared in a similar manner containing a final volume of 30 ml but no
selective agent.
Caffeine, when used, was added to layer 1, prior to pouring, at a final concentration of 1 mg m1-I. Solutions of
caffeine were made up in distilled water and membrane filtered (pore size 0.22 pm).
RESULTS
The relative sensitivities to FUV of five Uvs strains (CL77, CL89, CL90, CL104 and
CL 105) compared to strain CL7, the hypoxanthine-requiring auxotroph of S. clavuligerus
from which they were derived, are illustrated in Fig. 1.
Survival after FUV treatment of strain CL7 and the FUV-sensitive derivatives in the
presence and absence of caffeine is shown in Table 1. Caffeine (1 mg ml-') had previously
been shown to have no toxic effect on untreated spores. It can be seen that the presence of
caffeine also had no significant effect on the survival after FUV treatment of strains CL89,
CL90 and CL105. However, caffeine enhanced the killing effect of FUV in strains CL7,
CL77 and CL104.
The mutagenic responses of strains CL7, CL89, CL90 and CL105 in the presence and
absence of caffeine are shown in Table 2. Caffeine increased the frequency of FUV-induced
mutation to rifampicin resistance in strains CL7, CL90 and CL105. However, it had no effect
on the induced mutation frequency in strain CL89 and the magnitude of the effect was
significantly lower in strain CL105 than in CL7. Mutants CL89 and CL105 were
hypermutable relative to strain CL7.
In strain CL90, mutants were only obtained at very low FUV doses (up to 20 J m-*). The
mutagenic response of strain CL90 to X-rays was similar to that of strain CL7, whereas,
compared to strain CL7, strain CL90 was only slightly mutated by MMS. For example, an
MMS dose giving 170 acriflavine-resistant mutants per lo' survivors in strain CL7 gave only
10 per lo7survivors in strain CL90.
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Uvs mutants of Streptomyces clavuligerus
383
Fig. 1. FU survival curves of the progenitor strain CL7 (0)and Uvs strains CL
CL104 (U), CL90 (A)and CL89 (A).
Table 1. Percentage survival after FUV treatment of the wild-type strain (CL7) and FUVsensitive derivatives, grown in the absence and presence of caffeine
Percentage survival
A
f
3
Caffeine absent
Caffeine present
c
A
3 f
Strain
0
CL7
CL77
CL89
CL90
CL 104
CL105
100
100
100
100
100
100
FUV dose (J m-*):
5
10
15
20
0
98.0
90.2
44.6
51.6
69.6
67.0
95.5
80.3
4.7
24.4
16.4
34.3
100
100
100
100
100
100
97.2
95.6
73.0
74.7
80.9
85.1
92.9
84-7
10.0
35.2
44.7
43.1
\
FUV dose (J mP2):
5
10
15
79.7
90.2
68.7
77.1
44.2
87.3
20
78.5
69.2
11.0
29.2
4.1
37.1
81.3
81.7
39.5
56.3
13.2
67.0
50.1
55.6
5.3
24.8
2.0
28.1
Table 2. Frequency of FUV-induced mutation to rifarnpicin resistance for the wild-type strain
(CL7)and FUV-sensitive derivatives, grown in the absence and presence of caffeine
Mutants per lo7 survivors
I
Caffeine present
Caffeine absent
Strain
CL7
CL89
CL90
C L 105
A
A
f
0
0.8
0.5
0.5
1-6
\
FUV dose (J m-*):
5
10
15
2.9
74.0
0.20
18.0
6-1
118.0
4.4
94.0
14.0
43.0
9.3
124.0
20
18.0
50.0
16.0
184-0
(
\
0
0.9
0.4
0.3
2.5
FUV dose (J m-'):
5
10
15
91.0
50.0
10.4
72.0
166.0
95.0
13.2
200.0
198.0
54.0
34.4
400.0
20
212.0
32.0
35.2
820.0
In all experiments performed with strains CL77 or CL104 no detectable FUV-induced
mutants were obtained. Both strains showed significantly lower mutation frequencies than
strain CL7 when treated with the alkylating agent MMS. For example, an MMS dose giving
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3 84
G. SAUNDERS AND G. HOLT
255 acriflavine-resistant mutants per lo7survivors in strain CL7 gave only 71 in strain CL77
and 56 in strain CL104.
Mutants CL89, CL105 and CL77 showed a similar mutagenic response to NTG to that
observed in the wild-type strain CL7 (isolates CL90 and CL104 were not tested for their
response to NTG). All strains tested gave similar dose response curves with NTG treatment,
reaching a maximum mutation frequency of between 130 and 220 rifampicin-resistant
mutants per 107survivors.
DISCUSSION
Caffeine has been shown to inhibit excision repair in E. coli (Sideropoulos & Shankel,
1968) and, prior to this, Lieb (1961) demonstrated that FUV-induced mutation frequencies in
bacteria could be increased by caffeine. In our study, caffeine similarly affected mutation
frequencies in wild-type S . ctuvuligerus. The reduced effect of caffeine in mutant CL105 and
its apparent total ineffectiveness in mutant CL89 suggests that both these Uvs mutants are
excision-deficient, but possibly affected in different components of the excision repair system.
Hypermutability is a characteristic of excision-deficient strains of E. coli (Witkin, 1975).
The type of hypermutability observed in strain CL89 (where mutation frequency reaches a
peak and then declines) is identical to a measure of SOS activity in E . coZipolA mutants
(Witkin, 1976). The gene product of poZA is DNA polymerase I thought to effect the excision
and resynthesis steps of excision repair in E. coli. On the other hand, the pattern of
hypermutability observed in strain CL105 is similar to that found in E. coli exc mutants
deficient in the initial nicking or incision step of excision repair (Kondo et ul., 1970).
Fong & Bockrath (1979) suggested that caffeine inhibits the incision step of excision repair
in E. coli. The insensitivity of strain CL89 to caffeine is consistent with the suggestion that
CL89 is polymerase-deficient, and thus the effect of inhibiting the incision step in this strain is
negated by the absence of the next step, excision and/or resynthesis. Accordingly, strain
CL105 could be deficient in the initial incision step of excision repair and consequently exhibit
a reduced sensitivity to caffeine and an increased mutability towards FUV.
The phenotype of strain CL77 is such that it can readily be compared to lex mutants of E.
coli (Witkin, 1967). Strain CL77 and lex mutants have the common characteristic of yielding
no detectable mutants when treated with FUV and X-rays and significantly reduced mutation
frequencies when treated with MMS. Additionally, they are both mutated normally by NTG.
Strain CL104 was also similar in these respects, but it was far more sensitive to killing by
FUV than strain CL77 and for this reason we suggest that strain CL104 could carry a
mutation of the recA type found in E. coli. Thus, we suggest that strains CL77 and CL104
carry mutations at loci necessary for the operation of an error-prone pathway concerned with
mutation induction by FUV, X-rays and MMS and that NTG produces mutations through a
distinct pathway.
Strain CL90, whilst exhibiting a normal response to X-rays, showed a marked change in its
response to FUV and MMS. Unless one supposes that caffeine can increase the mutation
frequency in more than one way, which seems unlikely bearing in mind the results obtained
with strains CL89 and CL 105, the potentiation of FUV-induced mutation frequency
observed in this strain at low doses implies that the excision system for FUV damage is
operating. Further support for this is provided by the observation that the suggested
excision-deficient strains CL89 and CL105 are normal in their mutagenic response to MMS,
whilst CL90 is not. We therefore suggest that mutation induction by X-rays and FUV have
only some steps in common.
Harold & Hopwood (1970) isolated some 20 Uvs strains of Streptomyces coelicolor but
provided no evidence as to whether the mutagenic response of these strains differed from that
of the wild-type. Results obtained in our laboratory (Coleman & Holt, see Saunders et al.,
1982) have confirmed that in S . coelicolor certain uvs loci also confer hypermutability on a
strain.
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Uus mutants of Streptomyces clavuligerus
385
The ubiquity of DNA repair mechanisms in living organisms from bacteria to man
(Cleaver, 1968) is well known, and our studies indicate that S . clavuligerus has such repair
mechanisms and that their relationship to mutation induction appears to parallel closely the
relationships observed in E. coli.
We gratefully acknowledge the support of Beecham Pharmaceuticals in this work.
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